High-throughput knock-in coupling gene targeting with the HPRT minigene and Cre-mediated recombination.
Single nucleotide polymorphisms (SNPs) may influence protein function possibly contributing to phenotype; yet, for most SNPs their potential influence is unknown. Here, we present a technique in mouse embryonic stem cells that enables high-throughput knock-in (the placement of coding sequences adjacent to a specific endogenous promoter). Our methodology utilizes gene targeting with a combination of two selection cassettes (SAbetageo and the HPRT minigene) along with site-specific recombinases (Cre/loxP and FLP/FRT) to efficiently introduce multiple DNA sequences, including enhanced green fluorescent protein (eGFP), adjacent to the DNA topoisomerase 3beta (Top3beta) promoter. This technology enables rapid and efficient introduction of DNA sequences to a specific location and advances high-throughput analysis of many SNPs with control for expression and genetic background.
Kim
Tae Moon
TM
The Department of Molecular Medicine, The Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA.
Choi
Yong Jun
YJ
Ko
Jun Ho
JH
Hasty
Paul
P
D000818:Animals; D002678:Chimera / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism; D055879:Gene Knock-In Techniques; D018390:Gene Targeting / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000502:physiology; D011995:Recombination, Genetic
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; C045073:Cre recombinase; D019426:Integrases